The novel hematopoietic-specific SLFN14 endoribonuclease was initially discovered during the search for ribonucleases responsible for general translation control. Simultaneously, missense mutations were identified in a novel gene, SLFN14, in patients with a dominantly inherited form of thrombocytopenia, associated with excessive bleeding. Considering that SLFN14 is a key regulator of megakaryopoiesis and of structural development of platelets, we plan to use an integral approach to investigate the role of SLFN14 in platelet biogenesis. We will employ novel SLFN14 mouse models and inducible Pluripotent Stem Cell (iPSC) derived megakaryocytes (MKs) expressing SLFN14 patient mutations, in conjunction with biochemical, molecular biology, cellular biology techniques, and structural analysis via cryo-electron microscopy (cryo-EM). More specifically we will investigate how SLFN14 controls platelet formation and function by studying a platelet and megakaryocyte specific SLFN14 conditional knock-out mouse and knock-in mice with the K218E and K219N point mutations, with an initial phenotype analogous with human patients, alongside iPSC-derived megakaryocytes bearing patient SLFN14 mutations, created using CRISPR genome-editing, which we have already generated. To give further clues to the mechanism through which SLFN14 may regulate megakaryopoiesis, signaling, dense granule formation, platelet formation and activation, we will use RNA sequencing to analyze alterations in gene expression and the regulation of genes in MKs derived from our SLFN14-KO and SLFN14-KI mutant mice. Gene transcriptome and bioinformatic data analysis will define altered gene expression of upregulated/downregulated genes known or predicted to be associated with MK differentiation, maturation, platelet formation and function, as a result of SLFN14 mutation. Thermodynamics and kinetics of SLFN14-dependent RNA degradation in MKs and platelets derived from mutant mice and iPSCs will be studied. Identification of the SLFN14-specific cleavage sites within rRNA by footprinting analysis in the primary mouse platelets and iPSC-derived MKs will reveal sequence/structure cleavage specificity of the protein. To unveil whether SLFN14 disrupts the translational machinery by restricting cytoplasmic rRNA/tRNA/mRNA in iPSC-derived MKs and mouse platelets, polysome profiling and non-canonical amino acid labelling techniques will be utilized. Employment of selective inhibitors for the major degradation systems in iPSCs will reveal the degradation pathway underlying the autosomal dominant SLFN14-related thrombocytopenia. Mutational studies of SLFN14?s oligomerization motifs, endoribonuclease core, ribosomal binding and helicase domains coupled with the set of in vitro and in vivo assays will establish structure-function relationships of the protein. Binding partners of SLFN14 in MKs will be characterized. Structural analysis of SLFN14-associated 80S ribosomes and oligomeric forms of SLFN14 by cryo-EM will also be performed.